The present invention relates to the use of keratinocyte growth factor-2 (KGF-2) protein products to stimulate the proliferation, growth and differentiation of a variety of epithelial cells.
The complex process of tissue generation and regeneration is mediated by a number of protein factors sometimes referred to as soft tissue growth factors. These molecules are generally released by one cell type and act to influence proliferation of other cell types (Rubin et al. (1989), Proc. Nat""l. Acad. Sci. USA, 86:802-806). There are also some growth factors released from cells that themselves have the capacity to respond to such growth factors. Some soft tissue growth factors are secreted by particular cell types and influence the proliferation, differentiation, and/or maturation of responsive cells in the development of multicellular organisms (Finch et al. (1989), Science, 245:752-755). In addition to their roles in developing organisms, some soft tissue growth factors are significant in the continued health and maintenance of more mature systems. For instance, in mammals there are many systems where rapid cell turnover occurs. Such systems include the skin and the gastrointestinal tract, both of which are comprised of epithelial cells. Included within this group of soft tissue growth factors is a protein family of fibroblast growth factors (FGFs).
The fibroblast growth factor (FGF) family is now known to consist of at least fourteen members, namely FGF-1 to FGF-10 and homologous factors FHF-1 to FHF-4, which share a relatedness among primary structures: basic fibroblast growth factor, bFGF (Abraham et al. (1986), EMBO J., 5:2523-2528); acidic fibroblast growth factor, aFGF (Jaye et al. (1986), Science, 233:541-545); int-2 gene product, int-2 (Dickson and Peters (1987), Nature, 326:833); hst/kFGF (Delli-Bovi et al. (1987), Cell, 50:729-737 and Yoshida et al. (1987), Proc. Natl. Acad. Sci. USA, 84:7305-7309); FGF-5 (Zhan et al. (1988), Mol. Cell. Biol., 8:3487-3495); FGF-6 (Marics et al. (1989), Oncogene, 4:335-340); keratinocyte growth factor, KGF (Finch et al. (1989), Science, 24:752-755); hisactophilin (Habazzettl et al. (1992), Nature, 359:855-858); FGF-9 (Miyamoto et al. (1993), Mol. Cell Biol., 13(7):4251-4259); and fibroblast growth factor-10, also known as keratinocyte growth factor-2, KGF-2 (PCT patent application WO 96/25422), the disclosures of which are hereby incorporated by reference. More recently, four homologous factors (or xe2x80x9cFHFsxe2x80x9d) were identified from the human retina by a combination of random cDNA sequencing, searches of existing sequence databases and homology-based polymerase chain reactions (Smallwood et al. (1996), Proc. Natl. Acad. Sci. USA, 93:9850-9857). It has been proposed that FHF-1, FHF-2, FHF-3 and FHF-4 should be designated as FGF-11, FGF-12, FGF-13 and FGF-14, respectively, in accordance with the recommendation of the Nomenclature Committee (Coulier et al. (1997), Journal of Molecular Evolution, 44:43-56, the disclosure of which is hereby incorporated by reference).
WO 96/25422 describes the cloning, expression and purification of full-length (with signal sequence, residues Met1 to Thr36 of SEQ ID NO:2) and mature (without signal sequence, residues Cys37 to Ser208 of SEQ ID NO:2) KGF-2 in a bacterial expression system (e.g., E. coli) and eukaryotic expression systems (e.g., baculovirus and COS cells). This reference further teaches that KGF-2 might be useful to stimulate cell growth and proliferation for new blood vessel growth or angiogenesis, the prevention of hair loss, the healing of dermal wounds and the differentiation of muscle cells, nervous tissue, prostate cells and lung cells.
Much remains to be learned regarding KGF-2, including modifications which can be made thereto to generate variant(s) and derivatives which retain some or all of the biological activity of KGF-2. Generally, the effects of any specific amino acid change or chemical derivatization upon biological activity of a protein will vary depending upon a number of factors, including whether or not modifications affect the three-dimensional structure or the receptor binding region of the protein. As neither the three-dimensional structure nor the receptor binding region of KGF-2 has been published, the knowledge within the art does not permit generalization about the effects of specific amino acid modifications or chemical derivatization to KGF-2.
It is the object of this invention to provide variants and derivatives of KGF-2 that retain some or all of the biological activity of KGF-2.
The present invention is directed to KGF-2 protein product(s), as defined below. These KGF-2 protein product(s) have general applicability and may retain some or all of the biological activity of KGF-2.
In one aspect, a variant(s) of KGF-2 is produced by recombinant genetic engineering techniques. In an alternative embodiment, a variant(s) of KGF-2 is synthesized by chemical techniques, or a combination of the recombinant and chemical techniques A variant(s) of KGF-2 may be made in glycosylated or non-glycosylated form.
Yet another aspect of the present invention includes the various polynucleotides encoding a variant(s) of KGF-2. Each such nucleic acid sequence may be used in the expression of a variant(s) of KGF-2 in a eukaryotic or prokaryotic host cell. The polynucleotides may also be used in cell therapy or gene therapy applications.
A further aspect of the present invention involves vectors containing the polynucleotides encoding a variant(s) of KGF-2 operatively linked to amplification and/or expression control sequences.
A still further aspect of the present invention pertains to both prokaryotic and eukaryotic host cells containing recombinant polynucleotides encoding variant(s) of KGF-2.
In another aspect, the present invention further includes the recombinant production of a variant(s) of KGF-2 wherein recombinant host cells are grown in a suitable nutrient medium and wherein a variant(s) of KGF-2 expressed by the cells is, optionally, isolated from the host cells and/or the nutrient medium.
A still further aspect of the present invention includes KGF-2 protein(s), as defined below, attached to a water soluble polymer. For example, a variant(s) of KGF-2 may be conjugated to one or more polyethylene glycol molecules in order to improve pharmacokinetic performance by increasing the molecule""s apparent molecular weight.
Another aspect of the present invention includes pharmaceutical compositions containing a variant(s) of KGF-2, or chemical derivative(s) of KGF-2 protein(s). Typically, a variant(s) of KGF-2 may be formulated in association with pharmaceutically acceptable vehicles. A variety of other formulation materials may be used to facilitate the manufacture, storage, handling, delivery and/or efficacy of a variant(s) of KGF-2.
Yet another aspect relates to methods of modulating the growth and differentiation of epithelial cells. Specifically, a patient in need of stimulation (including cytoprotection, proliferation and/or differentiation) of epithelial cells will be administered a therapeutically-effective or prophylactically-effective amount of a variant(s) of KGF-2 and/or a chemical derivative of KGF-2 protein(s).
Additional aspects and advantages of the invention will be apparent to those skilled in the art upon consideration of the following description, which details the practice of the present invention.